Transforming growth factor-? (TGF?) regulates a wide variety of normal cellular processes including proliferation, survival, cell-matrix interaction, differentiation and plays a complex role during mammalian tumorigenesis. SnoN is a potent negative regulator of TGF? signaling through binding to and antagonizing the activity of the Smad proteins. It is a member of the Ski family of classically defined proto-oncogenes that when overexpressed, induces transformation of chicken and quail embryo fibroblasts. It is expressed in all adult cells and tissues at a low level but its expression is altered (up- or down-regulated) in many human cancer cells. Previous studies related to SnoN function mostly focused on its ability to promote oncogenic transformation in chicken embryo cells. Virtually nothing is known about its function in normal mammalian epithelial cells, and its role in mammalian tumorigenesis has not been well defined. The long-term goal of this proposal is to understand the function of SnoN and the SnoN/Smad interaction in regulation of cell proliferation, survival and senescence as well as mammalian tumorigenesis and to determine the molecular mechanisms underlying these processes. We will employ the mouse embryo fibroblasts (MEF) and MCF10A normal human mammary epithelial cell line to investigate the function of SnoN in normal mammalian cells. In an effort to determine the physiological significance of the SnoN/Smad interaction, we have isolated MEF from a strain of knock-in mice that express a mutant SnoN deficient in binding to the Smad proteins. These MEF cells display enhanced sensitivity to apoptotic stimuli and more interestingly, premature senescence, indicating that the SnoN/Smad interaction may regulate the apoptosis and senescence responses. We have also employed small-interference RNA approach in MCF10A cells and showed that SnoN promotes epithelial survival in a basement membrane-dependent manner. In this proposal, we would like to test the hypothesis that SnoN possess both anti-oncogenic and pro-oncogenic activities through regulation of cell senescence, survival and proliferation in both Smad-dependent and Smad-independent manner. The specific aims are: 1) To determine the molecular mechanism by which SnoN regulates cell senescence;2) To determine whether SnoN can function as a tumor suppressor through its ability to induce premature senescence;3) To determine the function of SnoN in normal human epithelial cells. These studies will allow us to understand the function of SnoN in normal mammalian cells and how deregulation of these activities facilitates tumorigenesis. PUBLIC HEALTH RELEVANCE: SnoN is a potent negative regulator of transforming growth factor-? (TGF-?) signaling, which play important but complex roles during mammalian tumorigenesis. It regulates many aspects of cellular functions including proliferation, differentiation, senescence and motility and contains both oncogenic and anti-oncogenic activities. Identification of the signaling pathways mediating each of these activities and deciphering the specificity of these pathways are crucial not only for understanding the mechanisms controlling malignant progression but also for development of novel cancer therapy that specifically targets the oncogenic activity of SnoN while preserving its anti-oncogenic activity.